Ethics declarations: The study conducted in accordance with the regulatory framework outlined by the "Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) 2012," as laid down under the "Prevention of Cruelty to Animals Act 1960" of the Indian Penal Code. The experimental protocols employed in this investigation received explicit approval from the Institutional Animal Ethics Committee (IAEC) i.e., 452/GO/S/03/CPCSEA- 25/07/2021 (Project code – P-1/2021/1-IAV/L34/3900/6100, Duration: 01-04-2021 to 31.03.2024). Reporting of in Vivo Experiments (ARRIVE) guidelines, ensuring the ethical conduct and reporting standards in the realm of animal experimentation.
Procedure followed for postbiotics collection:
A procedure11 was followed to isolate and collect postbiotic metabolites from probiotic bacteria (Lactobacillus acidophilus). The stock culture of probiotic (Lactobacillus acidophilus) procured from NCDC − 15 (National Collection of Dairy Cultures) at National Dairy Research Institute, India. Stock culture was revived twice by using de-Mann Rogosa Sharpe (MRS) broth (Hi Media – M369, granulated) with incubation at 30°C for 24 h, followed by spread plate inoculation of culture and incubation again at 30°C for 48 h. Each time, the incubation conditions were static. Then, a single colony was picked up and put into 10 ml MRS broth and incubated at 30°C for 24 hours. This will be followed by sub-culturing in 10 ml MRS broth and incubation at 30°C for 24 hours. From this culture, 1% (v/v) inoculum was added to the appropriate reconstitution media and incubated at 30°C for 24 hours. The bacterial cells were separated by centrifugation at 10,000 x g for 15 minutes, and the cell-free supernatant (CFS) was then collected by filtration through a syringe filter (Millex® Syringe Filters, 0.22 µm). The postbiotics (Supernatants) were collected and kept at 4°C until the feeding trial was conducted. The postbiotics produced were further put forwarded to various laboratory analysis such as pH, osmolarity, it’s anti-inflammatory, antioxidative and antimicrobial properties to evaluate its physiochemical properties12. Then the liquid postbiotics were sprayed on the feed everyday based upon the feed intake of the birds and mixed well before feeding to the respective treatments.
Management, diets, and experimental design of birds.
The present experimental study was conducted in experimental broiler farm at ICAR- Central Avian Research Institute, Bareilly, U.P. India. A total of 480 Day old CARI-Bro Dhanraja (slow growing-coloured broilers) chicks (straight run) were obtained from experimental hatchery of the institute and were randomly split up into six groups (completely randomized design). Each group was comprised of four replicates, and each replicates contained twenty chicks. Six different dietary treatments groups were, T1- Basal diet (BD) + 0.2%(v/w) MRS Broth/ uninoculated media; T2 - BD + Antibiotic (CTC@335mg/kg) {The antibiotic (CTC- Chlorotetracycline) was procured from Altron Biotech; T3 BD + Probiotic (Lactobacillus acidophilus − 1×106cfu/g); T4 - BD + Postbiotics @ 0.2% (v/w); T5 - BD + Postbiotics @ 0.4% (v/w) and T6 - BD + Postbiotics @ 0.6% (v/w). The total duration of the experiment was 42 days. Birds were fed with three different phases of diet such as pre-starter (0-14d), starter (15-28d) and finisher(29-42d) according to ICAR (2013) recommendations. The detailed outline of the feed ingredients employed in the study is given in the Table 1. The chicks were raised under an intensive, deep litter system. The birds were provided with ad libitum amounts of their respective feed and water. The birds have also administered with pertinent vaccinations in accordance with the standard vaccination schedule followed at the institute’s farm. The housing systems made as per the standard protocol, at initial for a week, birds were provided with 24 hrs of light, after that light duration has been decreased one hour each day until they had 18 hrs of light, which they continued to receive until the end of trial.
Table 1
Ingredient and chemical composition of basal diet (ICAR-2013)
Ingredients (%) | Pre-starter | Starter | Finisher |
Maize | 54.60 | 54.20 | 57.62 |
Soybean meal (CP, 44%) | 39.58 | 37.80 | 32.58 |
Rice bran oil | 02.12 | 04.24 | 05.86 |
Calcite | 01.54 | 01.52 | 01.73 |
Di-calcium phosphate | 0.90 | 0.95 | 1.10 |
Salt | 0.18 | 0.18 | 0.18 |
L – Lysine | 0.30 | 0.15 | 0.17 |
DL – Methionine | 0.30 | 0.28 | 0.27 |
Phytase | 0.015 | 0.015 | 0.015 |
Vitamin AD3EK1 | 0.014 | 0.014 | 0.014 |
B-complex vitamins2 | 0.015 | 0.015 | 0.015 |
Trace Minerals3 | 0.010 | 0.010 | 0.010 |
Coccidiostat | 0.01 | 0.01 | 0.01 |
Toxin binder | 0.05 | 0.05 | 0.05 |
Total | 100 | 100 | 100 |
Nutrient composition (%-)Calculated |
Crude protein | 22.65 | 21.65 | 19.70 |
Metabolizable energy (kcal/kg) | 3000 | 3125 | 3250 |
Calcium | 0.96 | 0.95 | 0.90 |
Available Phosphorus | 0.45 | 0.46 | 0.46 |
Lysine | 1.42 | 1.25 | 1.14 |
Methionine | 0.62 | 0.59 | 0.55 |
* | Calculated values |
1 | One gram of Vitamin AD3 EK supplement contained 82500 IU of Vitamin-A, 12000 IU of Vitamin-D3, 80 mg of Vitamin-E, and 10 mg of Vitamin-K. |
2 | One gram of B-complex supplement contained 8 mg of Vitamin-B1, 16 mg of Vitamin-B6, 80 mcg of Vitamin-B12, 120 mg of Niacin, 8 mg of Folic acid, 80 mg of Calcium -D-pantothenate and 86 mg of Calcium. |
3. | One gram of Trace mineral mixture contained 54 mg of Manganese, 52 mg of Zinc, 20 mg of Iron, 2 mg of Iodine and 1 mg of Cobalt. |
Zootechnical performance:
Weekly and cumulative production parameters including body weight, weight gain, feed intake, and feed conversion ratio (FCR) were methodically recorded for each treatment group. Each morning, precise quantities of feed were dispensed to the birds according to their respective dietary regimens, and the remaining feed was weighed the following day to determine total consumption. FCR was calculated on a weekly and cumulative basis using data from feed intake (FI) and body weight gain (BWG). Mortality among the experimental birds was monitored daily through meticulous observation and individual record-keeping.
Immune response
The immune status of the birds was evaluated in terms of humoral13 and cell mediated14 immune response, using standard methodologies. The weight of the immune organs was also recorded in this study.
Humoral immunity
Humoral immunity was evaluated by determining the antibody titre values against Newcastle disease (ND) in vaccinated birds. To facilitate this assessment, a booster dose of the La-Sota strain vaccine was administered to the birds via drinking water during their 3rd week of age. Antibody titre values were quantified using the haem-agglutination (HA) test procedure, conducted in U-bottom micro-titre plates.
Blood samples were collected from the jugular vein of healthy sheep using Alsever's solution as an anticoagulant. The collected blood was centrifuged at 2500 rpm for approximately 15 min after which the red blood cells were completely washed three times with phosphate buffer solution (PBS − 7.6) and the supernatant was discarded. Then, 1% v/v SRBC suspension was prepared in PBS and stored at 4ºC in refrigerator. 1.0 ml suspension of 1% v/v SRBC was injected intravenously to eight-birds/treatment on 28th day post hatch and the immunized birds were marked properly for easy identification. Blood (2 ml) from jugular veins of immunized birds were aseptically collected 5 days post immunization, i.e. on 34th day post hatch. The serum was separated and kept under frozen condition (-20 ºC), following which antibody titres to SRBC were analysed. A total of 98 U-bottom microtiter plates were utilized for the experiment. Each well received 50 µl of PBS as the initial solution. Subsequently, 50 µl of serum sample was added to the first well, followed by consecutive two-fold serial dilutions up to the 11th well. The 12th row served as the control. Following this, 50 µl of a 1% v/v suspension of sheep red blood cells (SRBC) in PBS was dispensed into each well. The microtiter plates were then placed in an incubator at 37ºC for 1 hour to facilitate incubation. After the incubation period, the plates were examined under bright light. The titration results were expressed as the log2 of the maximum dilution, which displayed irregular haemagglutination.
In-vivo cell mediated immune response (CMIR):
In this study, the cell-mediated immune response in birds was investigated by assessing their reaction to phytohemagglutinin type P (PHA-P), a protein variant of phytohemagglutinin-P (PHA-P) sourced from Hi-Media (TC226). On the 27th day post-hatch, each bird received an intradermal injection of 0.1 mL of PHA-P solution (1 mg/mL in PBS) between the 3rd and 4th interdigital spaces of the right foot. Concurrently, the left foot was injected with 0.1 mL of sterile phosphate-buffered saline (PBS) to serve as a control. Skin thickness measurements of both the right and left foot webs were taken using a micro-meter at both the time of injection (0 hours) and 24 hours post-injection of the mitogen. The in vivo response to PHA-P was quantified using the Foot Pad Index (FPI), computed as the difference between the swelling in the right foot and the left foot before and after the 24-hour injection period, and was reported in milli-meters.
Relative weight of lymphoid organs
Weights of lymphoid organs such as bursa of Fabricius, spleen and thymus (g) were recorded at 42 days of age and expressed as percentage of pre-slaughter live weight.
Serum assays
Biochemical serum profiling encompassing of protein, lipid, liver, kidney and mineral profiles was conducted utilizing commercial kits from Coral Clinical Systems (Tulip Diagnostics, Pvt. Ltd. Goa) along with spectrophotometer sourced from Bio-Rad Laboratories. At the age of 42 days, blood samples were randomly collected from 8 birds per treatment, into red-capped serum vials devoid of anticoagulants. After blood collection, the plasma samples were separated by centrifugation at 3000 rpm for 10 min and plasma was decanted into sterilized plastic vials, and then stored at -20°C for further use. The study employed various methods to determine serum biomarkers. Total protein estimation was performed using the Biuret method15, while the Bromocresol Green method was employed for albumin assessment16. Globulin estimation was derived using the formula: Globulin (g/dl) = Total Proteins (g/dl) – Albumin (g/dl), whereas the Albumin/Globulin Ratio (A/G Ratio) was calculated as Albumin (g/dl) divided by Globulin (g/dl). For the evaluation of plasma lipid profile, the enzymatic CHOD-PAP method was utilized for cholesterol determination, as per the methodology outlined by17; GPO/PAP method for triglycerides18. For plasma enzymes such as SGOT & SGPT the Reitman and Frankel method19, Kidney Profile, the Uricase method for uric acid20 and Modified Jaffe’s Kinetic Method for creatine21. For mineral profile, Calcium (OCPC method) 22 - Phosphorus (Molybdate U.V. method) 23, Calorimetric method for Sodium 24, Potassium25 & Chloride (thiocyanate method) were employed.
Gut Health
Caecal flora quantification:
On the 21st and 42nd day, samples of intestinal content (specifically caecal content), weighing approximately one gram, were meticulously collected from six birds per treatment and transferred into sterile containers to undergo microbiological analysis. Each gram of the sample was then diluted with 9 ml of 0.85% sterile saline solution, followed by homogenization and subsequent tenfold serial dilution. The diluted samples were plated in duplicate according to the standard procedure,27 onto selective media suitable for enumerating coliform, lactic acid bacteria, and total plate count. For the same MacConkey agar for coliform, MRS media for Lactic acid Bacteria and Hi-Veg Plate count agar for total plate count (Hi media) was utilized, and all media containing digesta samples (Spread plate method) were uniformly incubated at 37°C for 24 hours. Following incubation, colonies were selected and counted using a colony counter. The average count for each treatment was then calculated by multiplying the count obtained with the dilution factor and was expressed as log10 colony-forming units per gram (cfu/g) of the sample.
Morphometric analysis of jejunal tissue
For the histo-morphological assessment, samples of the jejunum were collected from six birds per treatment on days 21 and 42 of age. The jejunum samples were fixed in neutral buffered formalin and then processed for histology. Tissues were dehydrated using an ascending graded series of ethanol and embedded in paraffin wax. Using a microtome serial section of 5 µm thickness were cut from the jejunum samples. Four cross-sections of the jejunum per bird were then stained with Mayer’s Haematoxylin and Eosin (H&E) from Hi-Media Manufactures Ltd. Subsequently, morphometric and morphological analyses were conducted using light microscopy. The histological sections were examined under low magnification (10x), and parameters such as villus height, villus width, Villus height: Villus width ratio, crypt width and cryptal depth were measured using Zeiss Primo Star Software. In each cross-section, measurements were taken for ten villi and ten crypts per bird.
Jejunal tissue antioxidant activity
To ascertain the total antioxidant activity of the jejunal tissues, samples were procured from six birds per treatment on days 21 and 42 of the broilers' age. The assessment was conducted utilizing the Cayman Chemical Antioxidant Assay kit (709001) in strict accordance with the manufacturer's guidelines. The results were determined employing the specified formula. Antioxidant (mM) = [(Sample average absorbance) – (y-intercept) / Slope] x Dilution
Carcass characteristics
For carcass characteristics, the birds were slaughtered at the end of the trial, i.e., 42nd day, four birds from each replicate of the treatment group (20 birds/dietary treatment, n = 120) were randomly selected. The selected birds were fastened for about 12hrs, while having unrestricted access to drinking water. Afterwards, birds were euthanized to assess carcass traits and cut-up parts in percentage.
Statistical analysis
The statistical analysis was executed using Statistical Package for Social Sciences (SPSS) version 20.0, software. The data obtained from the conducted experiments underwent rigorous statistical analysis employing a one-way ANOVA framework, expressed as: Xij=µi+εij; Xij: The observed value for the dependent variable for the jth observation in the ith group; µi: Mean value for the ith group; εij: Represents the random error associated with the jth observation in the ith group. The group means (represented by µi) were compared using Tukey's multiple range test at a significance level of P ≤ 0.05 to determine if the differences between them were statistically significant.